This section shows and compare some metrics that indicate the quality of each sequencing run.

Polony numbers: total number of polonies. Each polony will yield one read1 and one read2 Base Qualities: percentage of bases that has >=30 (blue) or >=40 (black) base qualities Percentage assinged: percentage of reads that has been assigned to any sample Data Yield: the total volume of data that has been assigned to any sample

This sections plot the base qualities histograms.

Per base quality histogram plots the distribution of each inidividual base qualities.

Per read quality histogram plots the distribution of average base quality of each read

This section plots the base qualities by each sequencing cycle. You can choose to show either median, mean, percentage Q30 or percentage Q40 of each cycle. Read 1 and Read 2 are separated by a red dashed line, such that we can visualize R1 and R2 qualities in one plot.

This section shows numbers of some metrics that indicate the quality of each sa,[;e].

Polony numbers: total number of polonies assigned to this sample. Each polony yields one read1 and one read2

Percentage Q30: percentage of bases that has >=30 base quality (error rate <= 10^-3)

Percentage Q40: percentage of bases that has >=40 base quality (error rate <= 10^-4)

Average Base Quality: average base qualities across all bases

Yield: total volume of data that has been assigned to any sample

Read1 Average Length: the average length of Read 1

Read2 Average Length: the average length of Read 2

Group: A group tag for assigning colors in the plot. The default group of each sample will be their sequencing run name. To customize group tags, you can either

1) Set the project name when running bases2fastq. In this case the group tags will be project name.

2) Generate a csv file that has the columns "Run Name","Sample Name" and "Group". An easy way of generating this csv will be run multiqc with default grouping, copy this table into a csv file, and then change group name manually. Then move the csv file into any location under the analyzed directory.

This section plots the adapter content percentage by cycles

The heatmap is formatted in a similar way as fastqc per base sequence content. The differences are: 1) The pass/fail flag in the original fastqc module are replaced with a color reflecting the group tag of the samples 2) Read1 and Read2 is co-plotted and separate by a white band in the heatmap and a red dashed in each individual sample plot.

The following is a copy of helptext of the fastqc module: To enable multiple samples to be shown in a single plot, the base composition data is shown as a heatmap. The colours represent the balance between the four bases: an even distribution should give an even muddy brown colour. Hover over the plot to see the percentage of the four bases under the cursor.

To see the data as a line plot, as in the original FastQC graph, click on a sample track.

From the FastQC help:

Per Base Sequence Content plots out the proportion of each base position in a file for which each of the four normal DNA bases has been called.

In a random library you would expect that there would be little to no difference between the different bases of a sequence run, so the lines in this plot should run parallel with each other. The relative amount of each base should reflect the overall amount of these bases in your genome, but in any case they should not be hugely imbalanced from each other.

It's worth noting that some types of library will always produce biased sequence composition, normally at the start of the read. Libraries produced by priming using random hexamers (including nearly all RNA-Seq libraries) and those which were fragmented using transposases inherit an intrinsic bias in the positions at which reads start. This bias does not concern an absolute sequence, but instead provides enrichement of a number of different K-mers at the 5' end of the reads. Whilst this is a true technical bias, it isn't something which can be corrected by trimming and in most cases doesn't seem to adversely affect the downstream analysis.

This section plots the percentage of unidentified bases ("N" bases) by each sequencing cycle. Read 1 and Read 2 are separated by a red dashed line

This section plots the distribution of percentage GC in each reads (range: 0-100)